Molecular characterization of 52K protein of bovine adenovirus type 3

Bovine adenovirus (BAdV)-3 is a non-enveloped, icosahedral virus with a double-stranded DNA genome, and is being developed as a vector for vaccination of animals and humans. Expression of viral genes is divided into early, intermediate, and late phases. The late genes of BAdV-3 are grouped into seven families (L1 to L7) based on usage of common polyadenylation site(s). The L1 region of BAdV-3 encodes the 52K protein, a non-structural protein conserved among members of the family Adenoviridae. In human adenovirus (HAdV)-5, the 52K protein is involved in packaging of the viral DNA into the capsid. The N-terminal half of the protein has been proposed to mediate serotype specificity of DNA packaging. The objective of this study was to characterize the 52K protein of BAdV-3. DNA sequence analysis revealed that the BAdV-3 52K open reading frame encodes a protein of 370 amino acids rather than 331 amino acids as previously reported. Western blotting with anti-52K serum detected the expression of a 40kDa protein at 24 to 72 hrs post-infection. BAdV-3 52K localized predominantly to the nucleus in BAdV-3 infected cells and in transfected cells in the absence of other viral proteins. Analysis of mutant 52K proteins revealed that residues 102-110 were necessary but not sufficient for nuclear import. This suggests that residues upstream or downstream of the identified 52K nuclear localization signal (NLS) are required, or that the function of the NLS is dependent on its conformation within 52K. The nuclear import of 52K is significantly, but not completely, dependent on soluble factors, ATP, and temperature. A peptide competing for binding to importin beta and a peptide encoding the NLS of Ycbp80 were also able to inhibit nuclear import of 52K. However, a dominant negative mutant of Ran was unable to block 52K nuclear import. These results suggest that 52K uses a classical importin alpha/importin beta pathway for nuclear import. In support of this, a specific interaction between 52K and importin alpha-3 was detected. In addition, 52K was able to accumulate in the nucleus in the absence of soluble factors and ATP when the nuclear membrane was permeabilized with detergent. This suggests that, in addition to nuclear import by the importin alpha/importin beta pathway, 52K is able to accumulate in the nucleus by binding to nuclear components. A yeast two-hybrid system identified interactions between BAdV-3 52K and pV, pVI, pVII, and IVa2. However, only the interaction with pVII could be confirmed by GST pulldown. 52K and pVII also interact during BAdV-3 infection. An interaction between 52K and pVII has previously been shown in HAdV-5 infected cells. Mass spectrometry analysis of proteins co-precipitating with BAdV-3 52K identified a cellular protein, NFkB-binding protein (NFBP), which interacted with 52K. The interaction between NFBP and 52K was confirmed in vitro and in vivo. NFBP has been shown to be essential for ribosomal RNA (rRNA) processing. While NFBP is normally localized in the nucleolus, co-expression with 52K results in the redistribution of NFBP from the nucleolus to other parts of the nucleus. While this suggested that redistribution of NFBP by 52K could inhibit rRNA processing during BAdV-3 infection, we were unable to detect a difference in rRNA processing in cells expressing truncated or full-length 52K in the absence of other viral proteins. Since NFBP is a multi-functional protein, future experiments should focus on other possible biological functions of the interaction of NFBP with BAdV-3 52K